Renal Cell Carcinoma Discussion

9 LAFAYETTE COLLEGE Abstract Background: Patients with renal cell carcinoma (RCC), the most common type of kidney cancer (85% cases), have the lowest survival in the U.S. compared to all other urologic cancers. The most frequent RCC subtype is clear cell RCC (ccRCC, 75% cases) African American (AA) CCRCC patients have worse survival than European Americans (EAS). There are environmental lifestyle, and biological determinants of the racial disparity, with the tumor biology being the least understood. Genomic factors, like loss of heterozygosity (LOH), have been associated with poor cancer survival.

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LOH cccurs when a heterozygous cell losas one of its alleles at a specific locus, which increases cancer susceptibility One LOH mechanism, copy number loss (CNL)-LOM occurs when an allele is deleted or a portion of an allele is lost at a locus Hypothesis: CNL-LOH at aggressive tumor biology loci are population-specific Study Design: Discovery (TCGA) cohort data was downloaded, followed by clinical demographic characterization (n = 55 AAs n=458 EAS). Affymebix Genotyping Console 4.0 was used to generate Genome-Wide Human SNP Array 6.0 data. Partek Genomics Suite 7.0 LOH Workflow was used to perform paired LOH analysis (Threshold = 0.1). String v11.5 was used to determine known and predicted functional protein-protein interactions (PPI) Gene expression data in cBioPortal was downloaded to compare expression of scramblase genes by race Results: AAs had significant CNL-LOH on chromosomes 1, 11, and X, which was not observed in EAs. Both populations had significant CNL-LOH on chromosome 3. Further region-specific analyses of chromosome 3 showed that AAs had significant CNL-LOH on the 3q region, whille EAs exhibited significant CNL-LOH on 3p Collectively, 306 penes in 10 AA-enriched chromosome regions were impacted by CNL-LOH. Significant AA-PPI enrichment (P= 1.08-16) revealed greater biological inte interactions than expected and molecular functions in scramblase activity (P = 0.0382) Scramblase 5 was found to significantly interact with DIPKZA AAs had significantly lower expression of PLSCR4 and PLSCRS (P = 5.176-10, P= 2.130-10) Loss of heterozygosity in renal cell carcinoma from African American and European American patients Alex Bart and Khadijah A. Mitchell, PhD Lafayette College, Department of Biology, Easton, PA Conclusion: Population specific CNL-LOH at the global and chromosomal levels was identified in AAs with CCRCC Discussion: Scramblases are proapoptotic proteins and suspected tumor suppressors. We speculate CNL-LOH of scramblase genes can lead to loss of protein function, less apoptosis, greater disease progression, and decreased survival in AAS Future Directions: Future work will explore the association between coRCC patient survival by race, CNL-LOH status, and scramblase expression. Background Figure 1. African American (AA) kidney cancer patients have lower five-year survival than European American (EA) patients. 16 83.3% 14.6% Figure 2. Renal cell carcinoma (RCC) is the most common type of kidney cancer (-85% of cases), consisting of three subtypes. MAX Figure 3. AA RCC patients have worse survival than EAs for each RCC subtype Aims & Study Design Figure 4. Copy Number Loss-Loss of Heterozygosity (CNL-LOH) occurs either by simple deletion of one allele or deletion of a portion of an allele. 1. Imported 1113 CEL fies LOM Hypothesis and Specific Alms Hypothesis CNL LOH at aggressive tumor biology local are population-specific 1: Analyze CNL-LOM in AA and EA clear cell RCC (CCROC) 1. Imponied Denes im Interaction (PP) analysis based on Study Design Affymetrix Genotyping Canscle Workflow 1042 C O Age (years) Mean (SD) Female Male Stage (5) 1 2. QCod 1042 CEL fe in bounds Dena ithat is in bounds passed the OC cara Crecia 1: Smindt varaa by sa Requences Crema 2. Premove earth and sems with mr ara Copy number loss due to a portion d an avia being lest [I IV Ucknown Grade (%) + Partek Genomic Suite LOH Workflow 2 OCed 1047 OP les X Perfomed gmotyping sing dre Birdswd v2 algnet J 4 Unknown Vital Status (%) ^ & Dead Alive Unknown Fisher’s exact lest, Chi square lest, #Ilest unknown patients removed from significance testing 3. Performed paired LOH analysis on 910 Only tumor and normal pairs were included (n=43 AA and 406 [A paire) Results Table 1. Clinical and demographic information of ccRCC patients TCOA Discovery Compr 5 (9) 6 (11) 1(0) AA – 04 25 (46) 21 (38) 2 (4) 11 (20) 44 (30) 00 aported 1842 C-P Sim Clear Cell RCC AA (@-55) EA(-4) und and 4. Vinutlaed LOH results $9.9 (10.5) 60.6 (122) 37-81 26-90 STRING 153 (33) 305 (67) 217 (48) 49 (11) 112 (24) 78 (17) 20 458 10 (2) 18X (41) 181 (40) 74 (16) 5(0) 199 (15) 30 > 0.62 6.876 0.015 KBZJ Main Finding There were significant differences by sex, stage, grade and vital status In the TCGA Discovery Cohort AA chr with significant CNL-LOM Results Tabla 2. Chromosomes with significant CNL-LOH 3p25.3 3p24.1 2p221 p21.33 3:21.2 2p13 3013.32 3:21.3 34272 2 3:24 11p11.2 11p11.17 X011.22 X=25 AA Only 18:33 11p11.2 11p11.12 Xp11.22 Jp13 3q13.32 3021.3 3222 3424 3:213 3022.2 3024 AA only Main Findings AAs had significant CNL-LOH an Chromosomes 1, 11, and X, but EAs did not Both AAS and EAs had significant CNL-LOH on chromosome 3. CNL-LOH unique to AAs 81 7 53 13 Results Figure 5. Venn diagram of significant CNL-LOH on chromosome 3 cytobands 21 3 532 10 23 431 30241 371.33 3212 EA er with and CNL-LOM 0 0 0 10 0 3 0 Table 3. 306 candidate genes impacted by CNL-LOM across 10 cytobands 2 0 (A only 4 bay NO 1341 Genes Tumor Suppressor Genes Oncogenes 37 1 2 0 10 1 MOLL 3p28.2 3p28.1 621 3142 YO 3p25.2 3p25.1 3p24.2 3242 3:24.1 3222 22.1 3p21.31 3021.2 3p21.1 3143 Op 14.2 3p 14.1 CAL-LOH unique to AAS 0 1 5 0 O 3 71 7 12 18 8 43 10 Main Findings AAs mostly had significant CNL-LOH on the 3g region, while EAS exhibited significant CNL-LOH on 3p CNL-LOH impacted more tumor suppressor genes than oncogenes at 11p11.2, 3p13, and 3424. Results FASERS Figure 6. Scramblase protein-protein interactions P PLACES 11 Figure 7. Expression of scramblase genes by race Main Findings Four scramblases, tumor suppressor proteins, were functionaly envichet Scramblase 5 significantly interacted with DIPK2A AAs had significantly lower expression of PLSCR4 and PLSCRS. Conclusions & Discussion Conclusions •AAs had significant CNL-LOH on chromosomes 1, 11, and X • Both AAS and EAs had significant CNL-LOH on chromosome 1 with AAs having significant CNL-LOH on the 3q region while EA had significant CNL-LOH con the 3p region. • Of 306 genes impacted by CNL-LOH, scramblase proteins encoda by PLSCR genes were functionally enriched AAs had significantly lower expression of PLSCR4 and PLSCRS than EAS Discussion •AA scramblase tumor suppressor genes are more impacted by CNL-LOH in comparison to EA CORCC patients. Thus, CNE LOH scramblase genes in AAs la expected to lead to loss of protein function decreased apoptosis, greater disease progression, and worse survival. Future Directions Validate these findings in the CHTN Gesinger comert Perform survival analysis based on CNL-LOH status. Funding Source: Lafayette College Department of Biology9 LAFAYETTE COLLEGE Abstract Background: Patients with renal cell carcinoma (RCC), the most common type of kidney cancer (85% cases), have the lowest survival in the U.S. compared to all other urologic cancers. The most frequent RCC subtype is clear cell RCC (ccRCC, 75% cases). African American (AA) CCRCC patients have worse survival than European Americans (EAS). There are environmental, Mestyle, and biological determinants of the racial disparity, with the tumor biology being the least understood. Genomic factors, like loss of heterozygosity (LOH), have been associated with poor cancer survival, LOH occurs when a heterozygous cell loses one of its alleles at a specific locus, which increases cancer susceptibility. One LOH mechanism, copy number loss (CNL)-LOH. occurs when an allele is deleted or a portion of an allele is lost at a locus. Hypothesis: CNL-LOH at aggressive tumor biology loci are population-specific. Study Design: Discovery (TCGA) cohort data was downloaded, followed by clinical demographic characterization (n = 55 AAs, n = 458 EAs). Affymetrix Genotyping Console 4.0 was used to generate Genome-Wide Human SNP Array 6.0 data. Partek Genomics Suite 7.0 LOH Workflow was used to perform paired LOH analysia (Threshold = 0.1). String v11.5 was used to determine known and predicted functional protein-protein interactions (PPI). Gene expression data in cBioPortal was downloaded to compare expression of scramblase genes by race. Results: AAs had significant CNL-LOH on chromosomes 1, 11, and X, which was not observed in EAS. Both populations had significant CNL-LOH on chromosome 3. Further region-specific analyses of chromosome 3 showed that AAs had significant CNL-LOH on the 3q region, while EAs exhibited significant CNL-LOH on 3p. Collectively, 306 genes in 10 AA-enriched chromosome regions were impacted by CNL-LOH. Significant AA-PPI enrichment (P= 1.00-16) revealed greater biological interactions than expected and molecular functions in scramblase activity (P=0.0382). Scramblase 5 was found to significantly interact with DIPK2A AAs had significantly lower expression of PLSCR4 and PLSCRS (P= 5.176-10, P=2.13e-10). Conclusion: Population-specific CNL-LOH at the global and chromosomal levels was identified in AAs with ccRCC. Discussion: Scramblases are proapoptotic proteins and suspected tumor suppressors. We speculate CNL-LOH of scramblase genes can lead to loss of protein function, less apoptosis, greater disease progression, and decreased survival in AAs. Renal Cell Carcinoma Discussion

Future Directions: Future work will explore the association between ccRCC patient survival by race, CNL-LOH status, and scramblase expression. Background Figure 1. African American (AA) kidney cancer patients have lower five-year survival than European American (EA) patients. 91% 93.3% 74.7% EA Loss of heterozygosity in renal cell carcinoma from African American and European American patients Alex Bart and Khadijah A. Mitchell, PhD Lafayette College, Department of Biology, Easton, PA 14.6% Race National Cancer Indu, SEER Explorer, 2022 Lanmone & Physicpedia, 220 CI AA RCC Figure 2. Renal cell carcinoma (RCC) is the most common type of kidney cancer (-85% of cases), consisting of three subtypes 4040 Figure 3. AA RCC patients have worse survival than EAs for each RCC subtype. (x) penyaing angrepny weat-s t Ana M, Ungir Olgs 2020 ongo C American Cancer Soci 213 40 Aims & Study Design Figure 4. Copy Number Loss-Loss of Heterozygosity (CNL-LOH) occurs either by simple deletion of one allele or deletion of a portion of an allele. Copy number loss due to allele deletion LOM Hypothesis and Specific Aims Hypothesis: CNL-LOH at aggressive tumor biology lodi are population-specific. Alm 1: Analyze CNL-LOH in AA and EA clear cell RCC (ccRCC) patients from the TCGA Discovery Cohort 1. Imported 1113 CEL Mies 1. Imported 1042 CHP files Alm 2: Perform Protein-Protein Interaction (PPI) analysis based on AA-specific genes impacted by CNL-LOH. Sex (6) Female Male Study Design Affymetrix Genotyping Consols Workflow Age (years)# Mean (SD) 2. Oced Stage (%)+ I 11 FIX Copy number loss dun to a portion of an allele being lest 1042 CEL IV Unknown Grade (%) + in Bounds Data that is in-bounds passed the QC antena Criteria 1: Select variants by allele frequencies Crena 2. Remove variants and samples with missing data Partek Genomic Suite LOH Workflow 2. QCed 1042 CHP files LOW 3. Performed genotyping uning the Birdneed v2 algorithyn 2 3 4 Unknown Vital Status (%) ^ & Dead Alive Unknown ^ Fisher’s exact test,+ Chi square test, #lest & unknown patients removed from significance testing Only tumor and normal pairs were included (n=4 AA and 400 EA pairs) 3. Perfomed pared LOH analysis on 910 CHP Eles Results Table 1. Clinical and demographic information of ccRCC patients 28 (51) 27 (49) TCGA Discovery Cab 38 (69) 5 (9) 6 (11) 5 (9) 10) 25 (46) 21 (38) 2 (4) 3 (5) AA A-55 11 (20) 44 (80) 0 (0) 4. Generated and exported 1042 CHP les Clear Cell RCC AA(-55) EA(n-458) & Visualized LOH results Version 11.5 59.9 (10.5) 60.6 (122) 37-81 26-90 STRING 153 (33) 305 (67) n=459 217 (48) 49 (11) 112 (24) 78 (17) 2 (0) 10 (2) 188 (41) 181 (40) 74 (16) 5 (1) 159 (35) 295 (65) 3 (0) P 0.62 2016 0.016 6033 Main Finding There were significant differences by sex, stage, grade, and vital status in the TCGA Discovery Cohort. AA chr with significant CNL-LOH 1p33 3p26.3 3p25.3 3p24.1 3p22.1 3p21.33 3p21.2 3p13 3q13.32 3021.3 30222 3024 1a11.2 Results Table 2. Renal Cell Carcinoma Discussion

Chromosomes with significant CNL-LOH 11p11.12 Xp11.22 Xc25 AA Only 1p33 11p112 11p11.12 Xp11.22 3p13 3013.32 3p13 3q13.32 3:21.3 3422.2 3024 AA only 3421.3 39222 Main Findings AAs had significant CNL-LOH on Chromosomes 1, 11, and X, but EAs did not. Both AAS and EAs had significant CNL-LOH on chromosome 3. Results Figure 5. Venn diagram of significant CNL-LOH on chromosome 3 cytobands LA only 37 81 CNL-LOH unique to AAS 7 53 13 21 9 52 10 AA and EA 23 3p253 3241 3p23.1 371.33 EA chr with significant CNL-LOH 3p20.3 3p20.2 3p25.1 0 8 0 10 0 3 0 2 0 9211 Table 3. 306 candidate genes impacted by CNL-LOH across 10 cytobands #Genes Tumor Suppressor Genes Oncogenes 1 2 0 TO 1 4 1203 2243 421 $223 16211 1141 WILL 3p25.3 3p25.2 3p25.1 3p24.3 3p24.2 3p24.1 3p23 3p22.3 3p22.2 3p22.1 3021.33 3:21.32 3:21.31 3p21.2 3p21.1 3p14.3 3p14.2 3p14.1 CNL-LOH unique to AAs 0 0 1 5 0 TE “” INNBIND Other 35 71 7 33 12 13 8 43 10 19 Main Findings AAs mostly had significant CNL-LOH on the 3q region, while EAS exhibited significant CNL-LOH on 3p. CNL-LOH impacted more tumor suppressor genes than oncogenes al 11p11.2, 3p13, and 3024 PLECKI Results Figure 6. Scramblase protein-protein interactions 57 PLECRE Figure 7. Expression of scramblase genes by race 117 TA PLSCRA 3 PUSCES Main Findings Four scramblases, tumor suppressor proteins, were functionally enriched Scramblase 5 significantly interacted with DIPK2A AAs had significantly lower expression of PLSCR4 and PLSCR5. LA -2 Conclusions & Discussion ONTR/Gwainger Valictos Cohort Conclusions • AAs had significant CNL-LOH on chromosomes 1, 11, and X • Both AAS and EAs had significant CNL-LOH on chromosome 3 with AAs having significant CNL-LOH on the 3q region while Es had significant CNL-LOH on the 3p region. • Of 306 genes impacted by CNL-LOH, scramblase proteins encod by PLSCR genes were functionally enriched. • AAs had significantly lower expression of PLSCR4 and PLSCRI than EAs. Discussion • AA scramblase tumor suppressor genes are more impacted by CNL-LOH in comparison to EA coRCC patients, Thus, CNL-LOH scramblase genes in AAs is expected to lead to loss of protein function, decreased apoptosis, greater disease progression, and worse survival. Future Directions • Validate these findings in the CHTN/Geisinger cohort • Perform survival analysis based on CNL-LOH status. Funding Source: Lafayette College Department of Biology9 LAFAYETTE COLLEGE Abstract Background: Patients with renal cell carcinoma (RCC), the most common type of kidney cancer (85% cases), have the lowest survival in the U.S. compared to all other urologic cancera. The most frequent RCC subtype is clear cell RCC (ccRCC, 75% cases). African American (AA) CCRCC patients have worse survival than European Americans (EAS). There are environmental, lifestyle, and biological determinants of the racial disparity, with the tumor biology being the least understood. Genomic factors, like loss of heterozygosity (LOH), have been associated with poor cancer survival LOH occurs when a heterozygous cell loses one of its alleles at a specific locus, which increases cancer susceptibility. One LOH mechanism, copy number loss (CNL)-LOH. occurs when an allele is deleted or a portion of an allele is lost at a locus. Renal Cell Carcinoma Discussion

Hypothesis: CNL-LOH at aggressive tumor biology loci are population-specific. Study Design: Discovery (TCGA) cohort data was downloaded, followed by clinical demographic characterization (n = 55 AAs, n = 453 EAs). Affymetrix Genotyping Console 4.0 was used to generate Genome-Wide Human SNP Array 6.0 data. Partek Genomics Suite 7.0 LOH Workflow was used to perform paired LOH analyais (Threshold 0.1). String v11.5 was used to determine known and predicted functional protein-protein interactions (PPI). Gene expression data in cBioPortal was downloaded to compare expression of scramblase genes by race. Results: AAs had significant CNL-LOH on chromosomes 1, 11, and X, which was not observed in EAs. Both populations had significant CNL-LOH on chromosome 3. Further region-specific analyses of chromosome 3 showed that AAs had significant CNL-LOH on the 3q region, while EAs exhibited significant CNL-LOH on 3p. Collectively, 306 genes in 10 AA-enriched chromosome regions were impacted by CNL-LOH. Significant AA-PPI enrichment (P= 1.00-16) revealed greater biological interactions than expected and molecular functions in scramblase activity (P=0.0382) Scramblase 5 was found to significantly interact with DIPK2A. AAs had significantly lower expression of PLSCR4 and PLSCR5 (P= 5.17e-10, P=2.13e-10). Conclusion: Population-specific CNL-LOH at the global and chromosomal levels was identified in AAs with ccRCC Discussion: Scramblases are proapoptotic proteins and suspected tumor suppressors. We speculate CNL-LOH of scramblase genes can lead to loss of protein function, less apoptosis, greater disease progression, and decreased survival in AAs. Renal Cell Carcinoma Discussion

Criteria 1: Select variants by allele frequencies Criteria 2 Remove varams and samples with missing data Partak Genomic Suite LOH Workflow 3. Perfomed genotyping using the Extend 12 sigoriton 2 3 4 Unknown Vital Status (%) ^ & Dead Alive Unknown A Fisher’s exact test,+Chi square test, test & unknown patients removed from significance testing LOM 3. Perfomed parea LOH analysis on 910 CHP files Only tumor and normal pairs were included (49 AA and 408 EA pairs) 59.9 (10.5) 37-81 28 (51) 27 (49) 38 (69) Results Table 1. Clinical and demographic information of ccRCC patients 6 (11) 1(2) TOGA Discovery Cohert 25 (46) 21 (38) 2 (4) Clear Cell RCC AA (-55) EA(-45) 11 (20) 44 (80) 00 MA AW 85 & Generated and exported 4. Visualized LOH results Version 11.5 STRING 60.6 (122) 26-90 EA a=438 153 (33) 305 (67) 217 (45) (11) 112 (24) 78 (17) 2(0) 10 (2) 188 (41) 181 (40) 74 (16) 5 (1) 1.59 (35) 296 (65) 30 P 0.62 0.016 0.016 4033 Main Finding There were significant differences by sex, stage, grade, and vital status in the TCGA Discovery Cohort. AA chr with significant CNL-LOH 1:33 3626.3 325.3 Results Table 2. Chromosomes with significant CNL-LOH AA Only 1:33 11p11.2 11p11.12 Xo1122 3p13 3q13.32 3921.3 30222 3024 XC25 3p13 3:22.1 3p21.33 3p21.2 3q13.32 3421.3 3022.2 3024 3p13 3q13.32 3021.3 30222 3024 11p11.2 11p11.12 Xp11.22 XQ25 Main Findings AAs had significant CNL-LOH on Chromosomes 1, 11, and X, but EAs did not. Both AAs and EAs had significant CNL-LOH on chromosome 3. AA only CNL-LOH unique to AAS 81 7 53 13 Results Figure 5. Venn diagram of significant CNL-LOH on chromosome 3 cytobands EA only 21 9 52 10 AA and LA 23 3p24.3 3253 39241 39221 3p21 33 3021.2 0 8 0 10 0 Table 3. 306 candidate genes impacted by CNL-LOH across 10 cytobands 3 0 2 EA chr with significant CNL-LOH Mixt 0 Wil #Genes Tumor Suppressor Genes Oncogenes 1 37 4 4541 1:43 WAY 3p25.2 3:25.1 3:25.3 3:25.2 3p.25.1 3p24.3 3024.2 CNL-LOH unique to AAS 3p24.1 3p23 2 0 10 1 0 1 3:22.3 3:22.2 5 0 3p22.1 3p21.33 3p21.32 3:21.31 3p21.2 3:21.1 3p14.3 3014.2 3p14.1 0 Pas Lig ******** * Other 36 71 STIGIPLE 33 12 15 5 45 10 19 Main Findings AAs mostly had significant CNL-LOH on the 3q region, while EAS exhibited significant CNL-LOH on 3p. CNL-LOH impacted more tumor suppressor genes than oncogenes at 11p11.2, 3p13, and 3424 PUCKS Results Figure 6. Scramblase protein-protein interactions 57 La PLICK2 VEZ Ts Figure 7. Renal Cell Carcinoma Discussion

Expression of scramblase genes by race 3 B POCRA Main Findings Four scramblases, tumor suppressor proteins, were functionally enriched Scramblase 5 significantly interacted with DIPK2A AAs had significantly lower expression of PLSCR4 and PLSCRS. Conclusions & Discussion OTW Owainger Vadinin Cater AA Conclusions • AAs had significant CNL-LOH on chromosomes 1, 11, and X . Both AAS and EAs had significant CNL-LOH on chromosome 3 with AAs having significant CNL-LOH on the 3q region while EAs had significant CNL-LOH on the 3p region. • Of 306 genes impacted by CNL-LOH, scramblase proteins encoded LA BY 24 AUT by PLSCR genes were functionally enriched. •AAs had significantly lower expression of PLSCR4 and PLSCRS than EAs. Discussion . AA scramblase tumor suppressor genes are more impacted by CNL-LOH in comparison to EA ccRCC patients. Thus, CNL-LOH d scramblase genes in AAs is expected to lead to loss of protein function, decreased apoptosis, greater disease progression, and worse survival. Future Directions • Validate these findings in the CHTN/Geisinger cohort. • Perform survival analysis based on CNL-LOH status High CNL 4.0% of Bibion Funding Source: Lafayette College Department of Biology

Renal Cell Carcinoma Discussion